Beverages, notably beer, may be produced on the basis of barley—Hordeum vulgare, L.—which is a monocotyledonous crop plant cultivated in many parts of the world. Barley is economically important, both as a raw material for industrial products, including beer, and also as a source of animal feed.
In beer, as well as in many vegetables and foodstuffs—including tea, cocoa, milk, wines, spirits (such as rum), sweet corn, and numerous cooked vegetables—DMS adds prominent, generally beneficial odor and flavor notes to the product. However, a high level of DMS imparts a flavor that may be undesirable, usually described as “cooked sweet corn” or sometimes as “blackcurrant-like”.
Depending on beer type, DMS levels typically may be up to 144 μg/L and sometimes can reach as much as 150 ppb (150 μg/L), with said compound often contributing to undesirable “cooked vegetable” or “cabbage-like” flavors. However, a low level of the compound is sometimes desirable in lager beer as it may contribute to palate fullness and overall beer aroma; the sensory threshold is ˜25-50 ppb, for example around 30-45 μg/L (Meilgaard, 1982). The DMS flavour is generally not noticed when DMS levels are <10 ppb.
SMM, herein also denoted DMS precursor (DMSP), is synthesized in germinating barley kernels by the action of functional components of the SMM cycle (FIG. 1A). Here, the methionine (Met)-S-methyltransferase (MMT) enzyme catalyzes the transfer of a methyl group from S-adenosyl-methionine (AdoMet) to Met, forming SMM. The latter compound can in turn serve as methyl donor for Met synthesis from homocysteine (Hcy), a reaction catalyzed by the enzyme Hcy-S-methyltransferase (HMT). Although there is debate on the definitive role of MMT, it was initially proposed to prevent Met pools for protein synthesis from being depleted by an overshoot in AdoMet synthesis (Mudd and Datko, 1990). The SMM cycle also has been suggested to function in the long-distance sulfur transport in plants, with a major flux from Met to SMM in leaves, phloem transport of SMM, and reconversion of SMM to Met in developing kernels or other sink tissues (Bourgis et al., 1999). But later radiotracer experiments revealed that said cycle manages control of AdoMet levels rather than preventing depletion of Met (Ranocha et al., 2001). An alternative explanation on the physiological role of SMM in plants relates to the regulation of ethylene synthesis (Ko et al., 2004), principally with the ideas manifested through studies with recombinant 1-aminocyclopropane-1-carboxylate synthase from yeast of the genus Pichia. 
McElroy and Jacobsen (1995) have speculated that it may be possible to regulate SMM synthesis by using, for example, antisense technology. However, no guidance was provided on relevant target genes to antisense, but it was expected that the likelihood of a positive outcome was questionable as large reductions in SMM levels could be harmful to barley growth and development. Alternative solutions for obtaining lower level of SMM were not discussed by McElroy and Jacobsen (supra). In addition, as discussed in detail below, antisense technologies have not been successfully applied in barley to completely abolish gene expression.
Unfortunately, no methods are available for preparing transgenic barley plants that completely lack expression of a given protein. In general for barley, application of antisense techniques lead to transgenic plants still expressing some of the protein in question (see for example Robbins et al. 1998; Stahl et al., 2004; Hansen et al., 2007).
Also, effective methods for preparing specific mutations using chimeric RNA/DNA or site directed mutagenesis have not been developed for use in barley plants. In line with this, and despite intensive efforts, inventors of the present publication are not aware of any published example on successful oligonucleotide-directed gene targeting in barley. Although not pursued in barley, Iida and Terada (2005) note that oligonucleotide-directed gene targeting has been tested in maize, tobacco and rice—but in all cases with the herbicide-resistant gene acetolactate synthase (ALS) as a target. According to the conclusion by Iida and Terada (supra), it remains to be established whether the above-mentioned strategy, with appropriate modifications, is applicable to genes other than those directly selectable, such as the ALS genes. Targeted mutagenesis using zinc-finger nucleases represents another tool that potentially could allow future investigations in basic plant biology or modifications in crop plants (Durai et al., 2005; Tzfira and White, 2005; Kumar et al., 2006). Also in this case, mutagenesis has not been pursued or successfully applied in barley.
Nonetheless, barley mutants may be prepared by random mutagenesis using irradiation or chemical treatment, such as by treatment with sodium azide (NaN3). An example concerns barley kernels mutagenized through the use of NaN3, and subsequently screened for high levels of free phosphate in an effort to screen for low-phytate mutants (Rasmussen and Hatzack, 1998); a total of 10 mutants out of 2,000 screened kernels were identified. Although far from always possible, finding a particular mutant after NaN3 treatment is dependent on persistence and an effective screening method, and thus far from always successful.
A difficult part related to identification of promising molecular targets in traditional plant breeding concerns difficulties to ascertain which component(s) of a given biochemical pathway should be perturbed in order to produce the desired alteration of the system read out, and thus the ability to establish a useful screening method.
Beer production-related incubations at elevated temperatures—such as kiln drying of malt, or heating and boiling of wort—may induce chemical conversion of SMM to DMS. Due to inherent properties of DMS, particularly its boiling point of only 37° C.-38° C., a major part of the DMS formed during kilning and wort boiling may be lost to the atmosphere. At temperatures above ˜70° C., the volatility of DMS decreases to very low levels (Scheuren and Sommer, 2008), while manifesting conditions for further oxidation to dimethyl sulfoxide (DMSO). When hold time or vigor of wort boiling is inadequate to convert residual SMM, DMS may continue to form as the wort cools—with subsequent transfer into the beer.
Technological methods for reducing the level of DMS in beer have been developed. Thus, AU 38578/93 described a method of reducing DMS levels in malt, comprising steam treatment of said malt. In patent application US 2006/0057684 to Bisgaard-Frantzen, H. et al. was described brewing methods comprising heat treatment of mash at temperatures of 70° C. and above. And in U.S. Pat. No. 5,242,694 to Reuther, H. was described methods for preparing low carbohydrate beer, wherein the methods comprise extensive boiling of wort followed by carbon dioxide washing of said wort. However, all of the aforementioned treatments consume high levels of energy, and further they may alter the characteristics of malt or wort.